Diagnosis of cancer

ABSTRACT

A method is provided including obtaining an infrared (IR) spectrum of a blood plasma sample by analyzing the blood plasma sample by infrared spectroscopy, and based on the infrared spectrum, generating an output indicative of the presence of a solid tumor or a pre-malignant condition. Other applications are also described.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a U.S. national phase of PCT Application no.PCT/IL2012/000187 to Kapelushnik et al., filed May 10, 2012, whichpublished as WO 2012/153326 to Kapelushnik et al., and which claims thepriority of U.S. Provisional Patent Application 61/484,753 toKapelushnik et al., entitled, “Diagnosis of cancer,” filed May 11, 2011,which is incorporated herein by reference.

FIELD OF EMBODIMENTS OF THE INVENTION

Embodiments of the present invention relate generally to diagnosis andmonitoring of a disease, and particularly to methods for diagnosis andmonitoring of a malignant disease.

BACKGROUND

Analysis of certain markers (e.g., certain proteins, peptides, RNAmolecules) in a patient's circulation may be useful in detection and/ormonitoring of cancer. For example, studies have shown that analysis of apatient's blood plasma for certain oncofetal antigens, enzymes and/ormiRNA molecules may assist in diagnosis and prognosis of certain typesof cancer.

Fourier Transform Infrared (FTIR) spectroscopy is typically used toidentify biochemical compounds and examine the biochemical compositionof a biological sample. Typically, FTIR spectra are composed of severalabsorption bands, each corresponding to specific functional groupsrelated to cellular components such as lipids, proteins, carbohydratesand nucleic acids.

FTIR spectroscopy is used for analysis of various compounds in bloodplasma such as total proteins, creatinine, amino acids, fatty acids,albumin, glucose, fibrinogen, lactate, triglycerides, glycerol, urea,triglycerides, cholesterol, apolipoprotein and immunoglobulin.

Additionally, FTIR spectroscopy is commonly used to distinguish betweennormal and abnormal tissue by analyzing the changes in absorption bandsof macromolecules such as lipids, proteins, carbohydrates and nucleicacids. Additionally, FTIR spectroscopy may be utilized for evaluation ofcell death mode, cell cycle progression, and the degree of maturation ofhematopoietic cells.

SUMMARY OF EMBODIMENTS OF THE INVENTION

In some applications of the present invention, a method and system areprovided for the diagnosis and monitoring of multiple types of malignantneoplasms, particularly malignant solid tumors.

Additionally or alternatively, some applications of the presentinvention comprise diagnosis and monitoring of a pre-malignantcondition.

Typically, the method comprises analysis of blood plasma samples fromcancer patients by techniques of infrared (IR) spectroscopy, forexample, FTIR spectroscopy and/or microspectroscopy. As provided byapplications of the present invention, FTIR Optical Diagnosis Technology(FODT) allows analysis of biochemical changes in a blood plasma sampleof a patient which can indicate the presence of a solid tumor. Processessuch as carcinogenesis may trigger changes in a biochemical compositionof a body fluid of a patient, e.g., blood plasma. These changes aretypically represented by differences in the absorption and reflectionspectra when analyzed by FTIR spectroscopy techniques and compared toplasma samples from control individuals who do not suffer from amalignant solid tumor, e.g., healthy controls.

Accordingly, biochemical analysis of blood plasma samples obtained fromuntreated cancer patients and control individuals who do not suffer froma malignant solid tumor is conducted by FTIR microspectroscopy (FTIRMSP) techniques. Subsequently, the FTIR spectra (absorption and/orreflection) of blood plasma samples of the cancer patients are comparedto the FTIR spectra of blood plasma samples obtained from the controls.

The inventors have identified that the blood plasma samples obtainedfrom cancer patients suffering from a malignant solid tumor produce FTIRspectra that differ from those of the control individuals who do notsuffer from a malignant solid tumor, allowing distinguishing between thecancer patients and controls. Thus, some applications of the presentinvention can be used to diagnose cancer patients suffering from varioustypes of malignancies, particularly solid tumors. The distinction byFTIR spectroscopy between controls and patients suffering from solidtumors is typically performed based on analysis of blood plasma samplesand not of the actual tumor cells.

For some applications, a data processor is configured to analyze the IRspectrum, e.g., the FTIR spectrum, of the blood plasma sample of asubject and an output unit is configured to generate an outputindicative of the presence of a solid tumor, based on the infrared (IR)spectrum. Additionally, the data processor is typically configured tocalculate a second derivative of the infrared (IR) spectrum of the bloodplasma sample and, based on the second derivative of the infrared (IR)spectrum, to generate an output indicative of the presence of a solidtumor.

Additionally, some applications of the present invention allowdistinguishing between various types of solid tumors. For example, bloodplasma samples obtained from a cancer patient suffering from a certaintype of solid tumor produces an FTIR spectrum having a unique spectralpattern which is characteristic of the type of malignancy and distinctfrom spectra of other malignancy types.

For some applications, analysis by IR spectroscopy, e.g., FTIRspectroscopy of the biochemistry of blood plasma samples can be used forthe screening of large populations, aiding in the early detection ofcancer, including solid tumors. Infrared (IR) spectroscopy andmicrospectroscopy techniques (e.g., FTIR spectroscopy or FTIRMicrospectroscopy) as described herein are typically simple,reagent-free and rapid methods, suitable for use as screening tests forlarge populations. Early detection of cancer generally enables earlyintervention and treatment, contributing to a reduced mortality rate.

There is therefore provided in accordance with some applications of thepresent invention a method including:

obtaining an infrared (IR) spectrum of a blood plasma sample byanalyzing the blood plasma sample by infrared spectroscopy; and

based on the infrared spectrum, generating an output indicative of thepresence of a solid tumor or a pre-malignant condition.

For some applications, generating the output includes generating theoutput indicative of the presence of the solid tumor.

For some applications, analyzing the blood plasma sample by infrared(IR) spectroscopy includes analyzing the blood plasma sample by FourierTransformed Infrared (FTIR) spectroscopy, and obtaining the infrared(IR) spectrum includes obtaining a Fourier Transformed Infrared (FTIR)spectrum.

For some applications, analyzing the blood plasma sample by infrared(IR) spectroscopy includes analyzing the blood plasma sample by FourierTransformed Infrared microspectroscopy (FTIR-MSP).

For some applications, analyzing includes assessing a characteristic ofthe blood plasma sample at least one wavenumber selected from the groupconsisting of: 759±4 cm−1, 987±4 cm−1, 1172±4 cm−1, 1270±4 cm−1, 1283±4cm−1, and 1393±4 cm−1.

For some applications, analyzing includes assessing the characteristicat at least two wavenumbers selected from the group.

For some applications, analyzing includes assessing the characteristicat at least three wavenumbers selected from the group.

For some applications, analyzing includes assessing a characteristic ofthe blood plasma sample at at least one wavenumber selected from thegroup consisting of: 743±4 cm−1, 793±4 cm−1, 808±4 cm−1, 847±4 cm−1,850±4 cm−1, 895±4 cm−1, 950±4 cm−1, 961±4 cm−1, 963±4, 967±4 cm−1, 975±4cm−1, 997±4 cm−1, 1008±4 cm−1, 1030±4 cm−1, 1031±4 cm−1, 1048±4 cm−1,1120±4 cm−1, 1150±4 cm−1, 1159±4 cm−1, 1188±4 cm−1, 1205±4 cm−1, 1220±4cm−1, 1221±4 cm−1, 1255±4 cm−1, 1322±4 cm−1, 1326±4 cm−1, 1341±4 cm−1,1356±4 cm−1 1370±4 cm−1, 1372±4 cm−1, 1402±4 cm−1, 1415±4 cm−1, and1555±4 cm−1, 1595±4 cm−1, 1653±4 cm−1, 1681±4 cm−1.

For some applications, analyzing includes assessing the characteristicat at least two wavenumbers selected from the group.

For some applications, analyzing includes assessing the characteristicat at least three wavenumbers selected from the group.

For some applications, assessing the characteristic includes analyzing aband of the IR spectrum at at least one wavenumber selected from thegroup.

For some applications, the solid tumor includes a solid tumor in anorgan selected from the group consisting of: lung, pancreas, prostate,bladder, and gastrointestinal tract, and generating the output includesgenerating an output indicative of the presence of a solid tumor in anorgan selected from the group.

For some applications, the solid tumor includes breast cancer, andgenerating the output includes generating an output indicative of thepresence of breast cancer.

For some applications, analyzing the blood plasma sample includesobtaining a second derivative of the infrared (IR) spectrum of the bloodplasma sample.

For some applications, the blood plasma sample includes a dried bloodplasma sample and analyzing the blood plasma sample includes analyzingthe dried blood plasma sample.

For some applications, the infrared (IR) spectrum includes an absorptionspectrum and obtaining the infrared (IR) spectrum includes obtaining theabsorption spectrum.

For some applications, the infrared (IR) spectrum includes a reflectionspectrum and obtaining the infrared (IR) spectrum includes obtaining thereflection spectrum.

There is further provided in accordance with some applications of thepresent invention a method for monitoring the effect of an anti-cancertreatment on a subject undergoing anti-cancer treatment for a solidtumor, for use with a first blood plasma sample separated from blood ofthe subject that was obtained prior to initiation of the treatment and asecond blood plasma sample separated from blood of the subject that wasobtained after initiation of the treatment, the method including:

obtaining IR spectra of the first and second blood plasma samples byanalyzing the first and second blood plasma samples by IR spectroscopy;and

based on the IR spectra, generating an output indicative of the effectof the treatment.

For some applications, analyzing the first and second blood plasmasamples by IR spectroscopy includes analyzing the samples by FourierTransformed Infrared spectroscopy, and obtaining the IR spectra includesobtaining Fourier Transformed Infrared (FTIR) spectra.

For some applications, analyzing the first and second blood plasmasamples by infrared (IR) spectroscopy includes analyzing the first andsecond blood plasma samples by Fourier Transformed Infraredmicrospectroscopy (FTIR-MSP).

For some applications the method includes, obtaining an IR spectrum of athird blood plasma sample separated from blood of the subject that wasobtained following termination of the treatment, by analyzing the thirdblood plasma sample by IR spectroscopy.

For some applications, analyzing includes assessing a characteristic ofthe blood plasma sample at at least one wavenumber selected from thegroup consisting of: 759±4 cm−1, 987±4 cm−1, 1172±4 cm−1, 1270±4 cm−1,1283±4 cm−1, and 1393±4 cm−1.

For some applications, analyzing includes assessing the characteristicat at least two wavenumbers selected from the group.

For some applications, analyzing includes assessing the characteristicat at least three wavenumbers selected from the group.

For some applications, analyzing includes assessing a characteristic ofthe blood plasma sample at at least one wavenumber selected from thegroup consisting of: 743±4 cm−1, 793±4 cm−1, 808±4 cm−1, 847±4 cm−1,850±4 cm−1, 895±4 cm−1, 950±4 cm−1, 961±4 cm−1, 963±4, 967±4 cm−1, 975±4cm−1, 997±4 cm−1, 1008±4 cm−1, 1030±4 cm−1, 1031±4 cm−1, 1048±4 cm−1,1120±4 cm−1, 1150±4 cm−1, 1159±4 cm−1, 1188±4 cm−1, 1205±4 cm−1, 1220±4cm−1, 1221±4 cm−1, 1255±4 cm−1, 1322±4 cm−1, 1326±4 cm−1, 1341±4 cm−1,1356±4 cm−1 1370±4 cm−1, 1372±4 cm−1, 1402±4 cm−1, 1415±4 cm−1, and1555±4 cm−1, 1595±4 cm−1, 1653±4 cm−1, 1681±4 cm−1.

For some applications, analyzing includes assessing the characteristicat at least two wavenumbers selected from the group.

For some applications, analyzing includes assessing the characteristicat at least three wavenumbers selected from the group.

There is additionally provided in accordance with some applications ofthe present invention a method including:

obtaining an infrared (IR) spectrum of a blood plasma sample byanalyzing the blood plasma sample; and

based on the infrared spectrum, generating an output indicative of thepresence of a solid tumor or a pre-malignant condition.

For some applications, generating the output includes generating theoutput indicative of the presence of the solid tumor.

There is yet additionally provided in accordance with some applicationsof the present invention, a system for diagnosing a solid tumor,including:

a data processor, configured to analyze an infrared (IR) spectrum of ablood plasma sample of a subject; and

an output unit, configured to generate an output indicative of thepresence of a solid tumor, based on the infrared (IR) spectrum.

For some applications, the data processor is configured to calculate asecond derivative of the infrared (IR) spectrum of the blood plasmasample and, based on the second derivative of the infrared (IR)spectrum, to generate an output indicative of the presence of a solidtumor.

For some applications, the IR spectrum includes a Fourier TransformedInfrared (FTIR) spectrum, and the data processor is configured tocalculate a second derivative of the FTIR spectrum.

For some applications, the data processor is configured to analyze theinfrared (IR) spectrum by assessing a characteristic of the blood plasmasample at at least one wavenumber selected from the group consisting of:759±4 cm−1, 987±4 cm−1, 1172±4 cm−1, 1270±4 cm−1, 1283±4 cm−1, and1393±4 cm−1.

For some applications, the data processor is configured to analyze theinfrared (IR) spectrum by assessing the characteristic at at least twowavenumbers selected from the group.

For some applications, the data processor is configured to analyze theinfrared (IR) spectrum by assessing the characteristic at at least threewavenumbers selected from the group.

For some applications, the data processor is configured to analyze theinfrared (IR) spectrum by assessing a characteristic of the blood plasmasample at at least one wavenumber selected from the group consisting of:743±4 cm−1, 793±4 cm−1, 808±4 cm−1, 847±4 cm−1, 850±4 cm−1, 895±4 cm−1,950±4 cm−1, 961±4 cm−1, 963±4, 967±4 cm−1, 975±4 cm−1, 997±4 cm−1,1008±4 cm−1, 1030±4 cm−1, 1031±4 cm−1, 1048±4 cm−1, 1120±4 cm−1, 1150±4cm−1, 1159±4 cm−1, 1188±4 cm−1, 1205±4 cm−1, 1220±4 cm−1, 1221±4 cm−1,1255±4 cm−1, 1322±4 cm−1, 1326±4 cm−1, 1341±4 cm−1, 1356±4 cm−1 1370±4cm−1, 1372±4 cm−1, 1402±4 cm−1, 1415±4 cm−1, and 1555±4 cm−1, 1595±4cm−1, 1653±4 cm−1, 1681±4 cm−1.

For some applications, the data processor is configured to analyze theinfrared (IR) spectrum by assessing the characteristic at at least twowavenumbers selected from the group.

For some applications, the data processor is configured to analyze theinfrared (IR) spectrum by assessing the characteristic at at least threewavenumbers selected from the group.

There is still additionally provided in accordance with someapplications of the present invention, a computer program product foradministering processing of a body of data, the product including acomputer-readable medium having program instructions embodied therein,which instructions, when read by a computer, cause the computer to:

obtain an infrared (IR) spectrum of a blood plasma sample by analyzingthe blood plasma sample by infrared spectroscopy; and

based on the infrared spectrum, generate an output indicative of thepresence of a solid tumor.

The present invention will be more fully understood from the followingdetailed description of embodiments thereof, taken together with thedrawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-F are graphs representing FTIR absorption spectra and thesecond derivative of absorption spectra and analysis thereof, based onblood plasma from several cancer patients and controls, derived inaccordance with some applications of the present invention; and

FIG. 2 is a graph representing values of the second derivative ofabsorption spectra of blood plasma samples from cancer patients comparedto blood plasma samples from healthy controls, derived in accordancewith some applications of the present invention,

DETAILED DESCRIPTION OF THE EMBODIMENTS

Some applications of the present invention comprise diagnosis of a solidtumor by FTIR microspectroscopy (MSP) techniques. For some applications,FTIR Optical Diagnosis Technology (FODT) is used to diagnose a solidtumor based on biochemical properties of a blood plasma sample of asubject. Some applications of the present invention comprise obtaining ablood sample from a subject and analyzing plasma from the sample byFTIR-MSP techniques for the detection of a malignancy, specifically asolid tumor. Typically, blood plasma of a patient suffering from a solidtumor is identified as exhibiting FTIR spectra that are different fromFTIR spectra produced by blood plasma from a subject who does not sufferfrom a solid tumor (for some applications the control group may includesubjects suffering from a pathology that is not a solid tumor).Accordingly, some applications of the present invention provide a usefulmethod for the detection of cancer, specifically solid tumors. FTIRspectra of a blood plasma sample obtained from a cancer patient with asolid tumor generally reflect biochemical changes which occur in theblood plasma of the patient in response to the tumor.

For some applications, methods of the present invention are used todiagnose a type of solid tumor and/or a stage of the cancer.

For some applications, methods of the present invention can be used toprovide monitoring and follow up of cancer patients during and aftertreatment, e.g., chemotherapy treatment. Typically, changes in FTIRspectra of blood plasma samples of solid-tumor patients who areundergoing treatment can indicate biochemical changes in response to thetreatment. This biochemical information can provide insight into theeffect of treatment on the patient and/or the tumor.

Methods Used in Some Embodiments of the Present Invention

A series of protocols are described hereinbelow which may be usedseparately or in combination, as appropriate, in accordance withapplications of the present invention. It is to be appreciated thatnumerical values are provided by way of illustration and not limitation.Typically, but not necessarily, each value shown is an example selectedfrom a range of values that is within 20% of the value shown. Similarly,although certain steps are described with a high level of specificity, aperson of ordinary skill in the art will appreciate that other steps maybe performed, mutatis mutandis.

In accordance with some applications of the present invention, thefollowing methods were applied:

Obtaining Patient and Control Populations

All studies were approved by the local Ethics Committee of the SorokaUniversity Medical Center and conducted in accordance with theDeclaration of Helsinki. Qualified personnel obtained informed consentfrom each individual participating in this study.

The patient population included 28 cancer patients diagnosed with thefollowing primary tumors:

Breast (n=13), lung (n=4), pancreas (n=1), prostate (n=1) bladder (n=1),and gastrointestinal (n=7) and unknown origin (n=1) cancers.

The diagnosis of cancer was determined by clinical, surgical,histological, and pathologic diagnosis. The pathologic stage of thetumor was determined according to tumor-node-metastasis (TNM)classification, as described in “TNM Classification of MalignantTumours”, by Sobin LH. et al., 7th Edition, New York: John Wiley, 2009.A control group (n=42) included healthy volunteers who underwentdetailed clinical questioning, at the Soroka University Medical Centerand Ben-Gurion University.

Collection of Blood Samples

1-2 ml of peripheral blood was collected in 5 ml EDTA blood collectiontubes (BD Vacutainer® Tubes, BD Vacutainer, Toronto) from patients andhealthy controls using standard phlebotomy procedures. Samples wereprocessed within 2 hours of collection. It is to be noted that any othersuitable anticoagulant may be used in collection and processing of theblood samples.

Isolation of Plasma from Peripheral Blood Samples

Blood from cancer patients and healthy controls was diluted 1:1 inisotonic saline (0.9% NaCl solution). The diluted blood was appliedcarefully to Histopaque 1077 gradients (Sigma Chemical Co., St. Louis,Mo., USA) in 15 ml collection tubes, and centrifuged at 400 g for 30min.

To discard platelets and cell debris, the plasma was transferred to 1.5ml eppendorf tubes and centrifuged at 3,200 g for 10 min. Thesupernatant was transferred to a new eppendorf tube, and 0.5 μl ofplasma was deposited on a zinc selenide (ZnSe) slide. It is noted thatany other suitable slide may be used, e.g., reflection measurements maybe carried out using a gold slide. The slide was air dried for 1 hourunder laminar flow to remove water. The dried plasma was then subjectedto FTIR microscopy.

FTIR-Microspectroscopy

Fourier Transform Infrared Microspectroscopy (FTIR-MSP) and DataAcquisition Measurements were performed using the FTIR microscopeNicolet Centaurus with a liquid-nitrogen-cooledmercury-cadmium-telluride (MCT) detector, coupled to the FTIRspectrometer Nicolet iS10, OMNIC software (Nicolet, Madison, Wis.) usingOPUS software (Bruker Optik GmbH, Ettlingen, Germany). To achieve highsignal-to-noise ratio (SNR), 128 coadded scans were collected in eachmeasurement in the wavenumber region 700 to 4000 cm−1. The measurementsite was circular, with a diameter of 100 μm and spectral resolution of4 cm−1 (0.482 cm−1 data spacing). To reduce plasma sample thicknessvariation and achieve proper comparison between different samples, thefollowing procedures were adopted:

-   1. Each sample was measured at least five times at different spots.-   2. Analog to Digital Converter (ADC) rates were empirically chosen    between 2000 to 3000 counts/sec (providing measurement areas with    similar material density).-   3. The obtained spectra were baseline corrected using the rubber    band method, with 64 consecutive points, and normalized using vector    normalization in OPUS software as described in an article entitled    “Early spectral changes of cellular malignant transformation using    Fourier transformation infrared microspectroscopy”, by Bogomolny et    al., 2007. J. Biomed Opt. 12:024003.

In order to obtain precise absorption values at a given wavenumber withminimal background interference, the second derivative spectra were usedto determine concentrations of bio-molecules of interest. This method issusceptible to changes in FWHM (full width at half maximum) of the IRbands. However, in the case of biological samples, all samples (plasma)from the same type are composed of similar basic components, which giverelatively broad bands. Thus, it is possible to generally neglect thechanges in band FWHM, as described in an article entitled “Seleniumalters the lipid content and protein profile of rat heart: An FTIRmicrospectroscopy study”, by Toyran et al., Arch. Biochem. Biophys. 2007458:184-193.

Statistical Analysis:

Statistical analysis was performed using the student T-test.P-values<0.05 were considered significant. Statistical analysis wasperformed using STATISTICA software (STATISTICA, StatSoft, Inc., Tulsa,Okla.).

Artificial Neural Network Analysis:

Alyuda Neurolntelligence 2.2 (Alyuda Research Inc.) is a Neural NetworkSoftware for Classifying Data.

EXPERIMENTAL DATA

The experiments described hereinbelow were performed by the inventors inaccordance with applications of the present invention, and using thetechniques described hereinabove.

In a set of experiments, blood plasma from 42 healthy controls wasanalyzed by FTIR-MSP, and a typical FTIR-MSP spectral pattern wasestablished for control blood plasma. Additionally, blood plasma samplesfrom 28 cancer patients suffering from multiple types of solid tumorswere subjected to FTIR-MSP analysis and compared to the control FTIR-MSPspectral pattern. The blood plasma was obtained by preliminaryprocessing of the peripheral blood in accordance with the protocolsdescribed hereinabove with reference to isolation of plasma fromperipheral blood samples. The blood plasma samples were then analyzed byFTIR-MSP in accordance with the protocols described hereinabove withreference to FTIR-MSP.

Reference is made to FIGS. 1A-F, which are graphs representing FTIRabsorption spectra and the second derivative of absorption spectra andanalysis thereof, for blood plasma from 28 cancer patients and 42healthy controls, derived in accordance with some applications of thepresent invention.

FTIR-MSP analysis of blood plasma typically generated spectra in theregion of 4000-700 cm−1. The spectra are composed of several absorptionbands, each corresponding to specific functional groups of specificmacromolecules. FIGS. 1A-B show average FTIR-MSP spectra of blood plasmaof healthy controls and cancer patients in the regions of 3150-2830 cm−1(FIG. 1A) and 1800-700 cm−1 (FIG. 1B), after baseline correction andvector normalization. The main absorption bands are marked and themean±SEM is represented by the gray region around the average solid(control) and dotted (cancer) lines The absorption bands observed in theFTIR spectra shown in FIGS. 1A-B generally correspond to vibrations offunctional groups of molecules which are present in blood plasma. Forexample, proteins (e.g., albumin and globulins), nutrients (e.g.,glucose, amino acids, fatty acids, and monoglycerides) fibrinogen,electrolytes, solutes, hormones, enzymes, vitamins and other cellularcomponents; each has its own spectral fingerprint that together composethe entire spectra of the plasma sample. Each spectrum of a singleplasma sample represents the average of five measurements at differentsites for each sample.

As shown in FIGS. 1A-B, the FTIR-MSP spectra derived from analysis ofblood plasma from the cancer patients exhibited a different spectralpattern when compared to the FTIR-MSP spectra of blood plasma of healthycontrols.

Reference is made to FIG. 1A. Typically, the spectral region 3150-2830cm−1 contains absorption bands due to symmetric and asymmetric CH3 (at2959 cm−1, 2873 cm−1) and CH3 (at 2930 cm−1, 2856 cm−1) stretchingvibrations corresponding mainly to proteins and lipids respectively.Another absorption band located at ˜3060 cm−1 in this region typicallyis due to N—H stretching, and corresponds to Amide B. The absorption atthe band corresponding to Amide B was found to be significantly higher(p<2*10^−4) in the blood plasma samples of cancer patients when comparedto blood plasma from the healthy controls, as revealed by calculation ofthe area under the Amide B band (at 3014 cm−1 to 3110 cm−1) followingcut, baseline correction and Min-Max normalization at 2800 cm−1 to 3150cm−1.

Reference is made to FIG. 1B which shows a spectral region of 1800-700cm−1. Additionally, the insert in FIG. 1B shows a detailed view of the1400-900 cm−1 spectral region which has several overlapping bands whichcorrespond to multiple functional groups of plasma components. Thedetailed view of the 1400-900 cm−1 spectral region shows severaldifferences between blood plasma samples of cancer patients compared tohealthy controls, e.g., a reduction at 1400 cm−1 (containing COO⁻symmetric stretch) typically corresponding to protein.

Reference is made to FIGS. 1C and 1E. In order to increase accuracy andachieve effective comparison between the blood plasma samples of thecancer patients and the healthy controls, the second derivative of thebaseline-corrected, vector-normalized FTIR spectra was used. Results arepresented in FIGS. 1C and 1E. As shown, the second derivative spectralpattern of blood plasma samples from the cancer patients differedsignificantly from the FTIR-MSP spectral pattern of blood plasma of thehealthy controls. The main absorption bands are marked and the mean±SEMis represented by the gray area around the solid (controls) and dotted(cancer) lines.

Reference is made to FIGS. 1D and 1F, which are graphs showing ananalysis of the second derivative data shown in FIGS. 1C and 1E. Thegraphs in FIGS. 1D and 1F represent the variation between the secondderivative of the spectral pattern of blood plasma samples from thecancer patients and healthy controls, obtained in accordance withapplications of the present invention.

FIG. 2 shows a graph representing values of the second derivative ofabsorption spectra at wavenumbers A1-A21 of blood plasma samples fromcancer patients compared to blood plasma samples from healthy controls,derived in accordance with some applications of the present invention.Statistical analysis was performed and P-values are provided. As shown,the second derivative of blood plasma from the cancer patients differedsignificantly from the second derivative analysis of FTIR-MSP spectralpattern from blood plasma of healthy controls.

Table I lists the wavenumbers shown in FIG. 2. Typically, blood plasmasamples were analyzed by FTIR-MSP techniques using these wavenumbers todistinguish between healthy controls and cancer patients.

TABLE I Wavenumber (cm−1) ± 4 A1 743 A2 759 A3 847 A4 963 A5 967 A6 987A7 1030 A8 1150 A9 1172 A10 1205 A11 1221 A12 1270 A13 1283 A14 1326 A151356 A16 1372 A17 1393 A18 1555 A19 1595 A20 1653 A21 1681

The data obtain by analysis of the blood plasma samples may be furtheranalyzed by any suitable method known in the art, e.g., ArtificialNeural Network and/or Cluster Analysis, and/or Principal ComponentAnalysis, and/or Linear Discriminant Analysis (LDA) and/or Non LinearDiscriminant Analysis.

For example, data obtained in accordance with applications of thepresent invention was analyzed by artificial neural network (ANN).Several biomarkers shown in Table I which were statistically significant(p<0.05) were served as an input vector for the ANN analysis. Accordingto the ANN, 2 spectra out of 42 controls were rejected from theanalysis. These two spectra were suspected for improper samplepreparation. Twenty eight spectra were randomly selected for training,17 for validation and 23 for test. This procedure was repeated at leastten times (each time with different sets for training, test andvalidation) to confirm repeatability of the results. All of the ANNanalysis results presented high sensitivity and specificity of about 85%and 90%, respectively.

Reference is made to FIGS. 1 and 2.

It is further noted that the scope of the present invention includes theuse of only one wavenumber biomarker for detection and/or monitoring ofa solid tumor, as well as the use of two, three, four, or morewavenumbers.

Additionally, the scope of the present invention includes using any IRspectral feature or any feature derived from analysis of an IR spectralfeature (e.g., any type of peak analysis), to indicate the presence of asolid tumor.

It is also noted that the scope of the present invention is not limitedto any particular form or analysis of IR spectroscopy. For example, IRspectroscopy may include Attenuated Total Reflectance (ATR) spectroscopytechniques.

Although applications of the present invention are described hereinabovewith respect to spectroscopy, microspectroscopy, and particularly FTIRspectroscopy, the scope of the present invention includes the use ofanalysis techniques with data obtained by other means as well (forexample, using a monochromator or an LED, at specific singlewavenumbers, and/or FTIR imaging).

It is additionally noted that the scope of the present invention is notlimited to blood plasma and may apply to any treated or untreated bloodcomponent. For example, techniques and methods described herein mayalternatively be applied to blood serum.

It will be understood by one skilled in the art that aspects of thepresent invention described hereinabove can be embodied in a computerrunning software, and that the software can be supplied and stored intangible media, e.g., hard disks, floppy disks, a USB flash drive, orcompact disks, or in intangible media, e.g., in an electronic memory, oron a network such as the Internet.

It will be appreciated by persons skilled in the art that the presentinvention is not limited to what has been particularly shown anddescribed hereinabove. Rather, the scope of the present inventionincludes both combinations and subcombinations of the various featuresdescribed hereinabove, as well as variations and modifications thereofthat are not in the prior art, which would occur to persons skilled inthe art upon reading the foregoing description.

The invention claimed is:
 1. A method for indicating whether a subjecthas a gastrointestinal tract solid tumor, the method comprising:isolating, using a gradient, a blood plasma sample from a peripheralblood sample taken from the subject; drying, using a dryer, the bloodplasma sample of the subject; measuring an infrared (IR) spectrum of thedried blood plasma sample of the subject by analyzing the dried bloodplasma sample by infrared spectroscopy, and assessing a characteristicof the dried blood plasma sample at at least one wavenumber selectedfrom the group consisting of: 847±4 cm−1, 1150±4 cm−1, 1205±4 cm−1,1356±4 cm−1, and 1372±4 cm−1; using a data processor, comparing at theat least one wavenumber (a) the infrared spectrum of the dried bloodplasma sample of the subject to (b) an infrared spectrum obtained from adried plasma sample from a person without a solid tumor, to detect adifference between the infrared spectrum of the dried plasma sample ofthe subject and the infrared spectrum obtained from the dried plasmasample from the person without a solid tumor; and based on thecomparing, generating an output indicative of the presence of agastrointestinal tract solid tumor in the subject.
 2. The methodaccording to claim 1, wherein analyzing the dried blood plasma sample byinfrared (IR) spectroscopy comprises analyzing the dried blood plasmasample by Fourier Transformed Infrared (FTIR) spectroscopy, and whereinmeasuring the infrared (IR) spectrum comprises measuring a FourierTransformed Infrared (FTIR) spectrum.
 3. The method according to claim2, wherein analyzing the dried blood plasma sample by infrared (IR)spectroscopy comprises analyzing the dried blood plasma sample byFourier Transformed Infrared microspectroscopy (FTIR-MSP).
 4. The methodaccording to claim 1, wherein assessing the characteristic furthercomprises assessing a characteristic of the dried blood plasma sample atat least one wavenumber selected from the group consisting of: 759±4cm−1, 987±4 cm−1, 1172±4 cm−1, 1270±4 cm−1, 1283±4 cm−1, and 1393±4cm−1.
 5. The method according to claim 1, wherein analyzing comprisesassessing the characteristic at at least two wavenumbers selected fromthe group.
 6. The method according to claim 1, wherein assessing thecharacteristic further comprises assessing a characteristic of the driedblood plasma sample at at least one wavenumber selected from the groupconsisting of: 743±4 cm−1, 793±4 cm−1, 808±4 cm−1, 850±4 cm−1, 895±4cm−1, 950±4 cm−1, 961±4 cm−1, 963±4, 967±4 cm−1, 975±4 cm−1, 997±4 cm−1,1008±4 cm−1, 1030±4 cm−1, 1031±4 cm−1, 1048±4 cm−1, 1120±4 cm−1, 1159±4cm−1, 1188±4 cm−1, 1220±4 cm−1, 1221±4 cm−1, 1255±4 cm−1, 1322±4 cm−1,1326±4 cm−1, 1341±4 cm−1, 1370±4 cm−1, 1402±4 cm−1, 1415±4 cm−1, and1555±4 cm−1, 1595±4 cm−1, 1653±4 cm−1, 1681±4 cm−1.
 7. The methodaccording to claim 1, wherein assessing the characteristic comprisesanalyzing a band of the IR spectrum at the at least one wavenumberselected from the group.
 8. The method according to claim 1, whereinanalyzing the dried blood plasma sample comprises obtaining a secondderivative of the infrared (IR) spectrum of the dried blood plasmasample.
 9. A system for indicating whether a subject has agastrointestinal tract solid tumor, the system comprising: a gradientconfigured to isolate a blood plasma sample from a peripheral bloodsample of the subject; a dryer configured to dry the blood plasmasample; a data processor, configured (i) to analyze an infrared (IR)spectrum of the dried blood plasma sample of the subject by assessing acharacteristic of the dried blood plasma sample at at least onewavenumber selected from the group consisting of: 847±4 cm−1, 1150±4cm−1, 1205±4 cm−1, 1356±4 cm−1, and 1372±4 cm−1, and (ii) to compare atthe at least one wavenumber (a) the infrared spectrum of the dried bloodplasma sample of the subject to (b) an infrared spectrum obtained from adried plasma sample from a person without a solid tumor, to detect adifference between the infrared spectrum of the dried plasma sample ofthe subject and the infrared spectrum obtained from the dried plasmasample from the person without a solid tumor; and an output unit,configured to generate an output indicative of the presence of agastrointestinal tract solid tumor in the subject, based on thecomparing.
 10. The system according to claim 9, wherein the dataprocessor is configured to calculate a second derivative of the infrared(IR) spectrum of the dried blood plasma sample and, based on the secondderivative of the infrared (IR) spectrum, to generate an outputindicative of the presence of the gastrointestinal tract solid tumor.11. The system according to claim 10, wherein the IR spectrum includes aFourier Transformed Infrared (FTIR) spectrum, and wherein the dataprocessor is configured to calculate a second derivative of the FTIRspectrum.
 12. The system according to claim 9, wherein the dataprocessor is further configured to analyze the infrared (IR) spectrum byassessing a characteristic of the dried blood plasma sample at at leastone wavenumber selected from the group consisting of: 759±4 cm−1, 987±4cm−1, 1172±4 cm−1, 1270±4 cm−1, 1283±4 cm−1, and 1393±4 cm−1.
 13. Thesystem according to claim 9, wherein the data processor is configured toanalyze the infrared (IR) spectrum by assessing the characteristic at atleast two wavenumbers selected from the group.
 14. The system accordingto claim 9, wherein the data processor is further configured to analyzethe infrared (IR) spectrum by assessing a characteristic of the driedblood plasma sample at at least one wavenumber selected from the groupconsisting of: 743±4 cm−1, 793±4 cm−1, 808±4 cm−1, 850±4 cm−1, 895±4cm−1, 950±4 cm−1, 961±4 cm−1, 963±4, 967±4 cm−1, 975±4 cm−1, 997±4 cm−1,1008±4 cm−1, 1030±4 cm−1, 1031±4 cm−1, 1048±4 cm−1, 1120±4 cm−1, 1159±4cm−1, 1188±4 cm−1, 1220±4 cm−1, 1221±4 cm−1, 1255±4 cm−1, 1322±4 cm−1,1326±4 cm−1, 1341±4 cm−1, 1370±4 cm−1,1402±4 cm−1, 1415±4 cm−1, and1555±4 cm−1, 1595±4 cm−1, 1653±4 cm−1, 1681±4 cm−1.
 15. A computerprogram product for administering processing of a body of data, theproduct comprising a computer-readable medium having programinstructions embodied therein, which instructions, when read by acomputer, cause the computer to: measure an infrared (IR) spectrum of adried blood plasma sample isolated from a peripheral blood sample of asubject, by analyzing the dried blood plasma sample by infraredspectroscopy; assess a characteristic of the dried blood plasma sampleat at least one wavenumber selected from the group consisting of: 847±4cm−1, 1150±4 cm−1, 1205±4 cm−1, 1356±4 cm−1, and 1372±4 cm−1; compare atthe at least one wavenumber (a) the infrared spectrum of the dried bloodplasma sample of the subject to (b) an infrared spectrum obtained from adried plasma sample from a person without a solid tumor, to detect adifference between the infrared spectrum of the dried plasma sample ofthe subject and the infrared spectrum obtained from the dried plasmasample from the person without a solid tumor; and based on thecomparison, generate an output indicative of the presence of agastrointestinal tract solid tumor in the subject.